User:Allison K. Alix/Notebook/Thesis Research/2013/05/14: Difference between revisions
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== | ==Objectives== | ||
* | |||
* React thiol-DNA/ThT/AuNP at 150:1 concentrations of thiol-DNA:AuNP | |||
* Run 2% agarose gel on thiol-DNA/ThT/AuNP synthesized on [[User:Allison K. Alix/Notebook/Thesis Research/2013/04/23|04/23/2013]] | |||
* Take absorbance and fluorescence measurements of thiol-DNA/ThT before and after attaching AuNP | |||
==Procedures== | |||
* New AuNP synthesized is 29.67nM with a diameter of 10nm | |||
Previous concentrations used: | |||
{| class="wikitable" | |||
|- | |||
! !! Initial !! Final | |||
|- | |||
| thiol-DNA || 5.82μM || 727.5nM | |||
|- | |||
| ThT || 5μM || 625nM | |||
|- | |||
| AuNP || 19.4nM || 9.7nM | |||
|} | |||
To be used: | |||
{| class="wikitable" | |||
|- | |||
! !! Initial !! Final | |||
|- | |||
| thiol-DNA || 17.8μM || 2.225μM | |||
|- | |||
| ThT || 20μM || 2.5μM | |||
|- | |||
| AuNP || 29.67nM || 14.835nM | |||
|} | |||
'''Calculations:''' | |||
''Preparing 17.8μM thiol-DNA'' | |||
original concentration of thiol-DNA- 53.1μM | |||
53.1μM (x) = (17.8μM)(250μL) | |||
x= 83.8μL in 166.2μL buffer | |||
''Preparing 20μM ThT'' | |||
original conc ThT= 5mM | |||
5mM (x) = (0.02mM)(10mL) | |||
x=40μL in 9960μL H<sub>2</sub>O | |||
'''Part 1: hybridizing thiol-DNA with ThT''' | |||
1) Mix 250μL 17.8μM thiol-DNA with 250μL 20μM ThT | |||
* NEW concentrations: 8.9μM thiol-DNA and 10μM ThT | |||
2) Heat at 75°C for ~25min | |||
3) Cool to room temperature | |||
4) Take UV-Vis/Fluorescence measurements of thiol-DNA/ThT sample | |||
'''Part 2: Hybridizing thiol-DNA/ThT with AuNP''' | |||
1) Mix 250 μL 8.9μM thiol-DNA/10μM ThT solution with 250μL 4%TEA containing 100mM DTT | |||
* NEW concentrations: 4.45μM thiol-DNA/5μM ThT | |||
2) Extract DTT using 4 2mL aliquots of ethyl acetate | |||
3) Mix 232.4μL of thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL sodium citrate buffer | |||
* NEW concentrations: 2.06μM thiol DNA, 2.324μM ThT, 13.79nM AuNP | |||
'''Part 3: Gel Electrophoresis (2% Agarose)''' | |||
Prepare the following: | |||
1) 5μL 100bp ladder with 1μL loading buffer | |||
2) 5μL thiol-DNA/ThT/AuNP with 1μL loading buffer | |||
3) 5μL supernatant 1 with 1μL loading buffer | |||
4) 5μL supernatant 2 with 1μL loading buffer | |||
5) 5μL supernatant 3 with 1μL loading buffer | |||
==Data== | |||
[[Image:Pic_of_abs.png]] | |||
[[Image:Pic_of_fluor.png]] | |||
[[Image:AliDNAThTAuNPGel.png]] | |||
==Observations== | |||
In the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point. | |||
* Note that for the gel electrophoresis, if using the blue/orange loading dye, allow the gel to run until the lightest blue dye is 3/4 of the way across, this should be the dye that travels the slowest. (Overall took about ~2 hours) | |||
No DNA streaks were visible on the gel. The sample in the first well was the 100bp ladder which did show a substantial streak. I was expecting the supernatants (wells 3-5) to show a little streak if any loose DNA was present and I was not expecting the actual concentrated sample (well 2) to exhibit a streak. | |||
Tomorrow I will try running a gel with freshly prepared thiol-DNA/ThT/AuNP | |||
Revision as of 09:04, 15 May 2013
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Objectives
Procedures
Previous concentrations used:
To be used:
Calculations: Preparing 17.8μM thiol-DNA original concentration of thiol-DNA- 53.1μM 53.1μM (x) = (17.8μM)(250μL) x= 83.8μL in 166.2μL buffer Preparing 20μM ThT original conc ThT= 5mM 5mM (x) = (0.02mM)(10mL) x=40μL in 9960μL H2O
1) Mix 250μL 17.8μM thiol-DNA with 250μL 20μM ThT
2) Heat at 75°C for ~25min 3) Cool to room temperature 4) Take UV-Vis/Fluorescence measurements of thiol-DNA/ThT sample Part 2: Hybridizing thiol-DNA/ThT with AuNP 1) Mix 250 μL 8.9μM thiol-DNA/10μM ThT solution with 250μL 4%TEA containing 100mM DTT
2) Extract DTT using 4 2mL aliquots of ethyl acetate 3) Mix 232.4μL of thiol-DNA/ThT with 232.4μL 29.67nM AuNP and 35.2μL sodium citrate buffer
Part 3: Gel Electrophoresis (2% Agarose) Prepare the following: 1) 5μL 100bp ladder with 1μL loading buffer 2) 5μL thiol-DNA/ThT/AuNP with 1μL loading buffer 3) 5μL supernatant 1 with 1μL loading buffer 4) 5μL supernatant 2 with 1μL loading buffer 5) 5μL supernatant 3 with 1μL loading buffer Data
ObservationsIn the absorbance spectrum, a peak exhibited at 436nm can be attributed to the ThT. The fluorescence spectrum shows an intensity well above what the instrument can measure, but one can infer that there is a peak around ~500nm, which is where ThT, fluoresces. The concentrations of DNA and ThT will decrease once the AuNP are attached. This being said, the fluorescence intensity should be measurable at that point.
No DNA streaks were visible on the gel. The sample in the first well was the 100bp ladder which did show a substantial streak. I was expecting the supernatants (wells 3-5) to show a little streak if any loose DNA was present and I was not expecting the actual concentrated sample (well 2) to exhibit a streak. Tomorrow I will try running a gel with freshly prepared thiol-DNA/ThT/AuNP
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